抑制自噬对根尖乳头干细胞成骨分化水平的影响

目的探讨抑制自噬对根尖乳头干细胞成骨分化水平的影响。方法5、10 ng/mL 肿瘤坏死因子-α（TNF-α）分别处理根尖 乳头干细胞（SCAPs），对照组不做处理，检测自噬相关蛋白LC3-II表达水平，GFP-LC3质粒转染并检测细胞内GFP-LC3数目， 吖啶橙染色检测酸性囊泡情况。TNF-α，TNF-α+3-MA分别处理SCAPs，检测LC3-II 的表达水平，GFP-LC3 质粒转染并检测 GFP-LC3数目，CCK-8法检测细胞活力，流式细胞仪检测细胞凋亡。TNF-α、TNF-α+3-MA分别处理SCAPs，对照组不做处理， 诱导成骨向分化，qRT-PCR检测分化第3、7、14天时成骨相关基因碱性磷酸酶（ALP）、骨涎蛋白（BSP）、骨钙素（OCN）的表达。 结果TNF-α可诱导SCAPs自噬活化：TNF-α组LC3-II/β-actin水平较对照组升高且具有浓度依赖性（P<0.05），TNF-α组GFP-LC3 数目较对照组升高且具有浓度依赖性（P<0.05），TNF-α组酸性囊泡较对照组增多。3-MA可抑制TNF-α诱导的SCAPs自噬活 化：TNF-α+3-MA组LC3-II/β-actin水平及细胞内GFP-LC3数目较TNF-α组显著降低（P<0.05），并下调细胞活力（P<0.05），上调 细胞凋亡水平（P<0.05）。抑制自噬导致SCAPs成骨分化的抑制：TNF-α+3-MA组ALP、BSP表达量在分化第3、7、14 天均较 TNF-α组降低（P<0.05），OCN的表达量在第3、7天较TNF-α组降低（P<0.05）。结论TNF-α可诱导SCAPs自噬水平的活化；自噬 可能对TNF-α作用下的SCAPs起细胞保护作用，对抗细胞凋亡；自噬的抑制将下调TNF-α作用下SCAPs的成骨向分化水平，提 示自噬在TNF-α作用下SCAPs的成骨分化过程中具有重要作用。

Inhibition of autophagy suppresses osteogenic differentiation of stem cells from apical papilla

Objective To investigate the effects of autophagy on osteogenic differentiation of stem cells from the apical papilla (SCAPs) in the presence of tumor necrosis factor-α (TNF-α) stimulation in vitro. Methods SCAPs treated with TNF-α (0, 5, and 10 ng/mL) with or without 5 mmol/L 3-MA were examined for the expression of autophagy marker LC3- II using Western blotting. The cells were transfected with GFP-LC3 plasmid and fluorescence microscopy was used for quantitative analysis of intracellular GFP-LC3; AO staining was used to detect the acidic vesicles in the cells. The cell viability was assessed with CCK- 8 assays and the cell apoptosis rate was analyzed using flow cytometry. The cells treated with TNF-α or with TNF-α and 3-MA were cultured in osteogenic differentiation medium for 3 to 14 days, and real- time PCR was used to detect the mRNA expressions of osteogenesis-related genes (ALP, BSP, and OCN) for evaluating the cell differentiation. Results TNF-α induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF-α-induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs (P<0.05). Compared with the cells treated with TNF-α alone, the cells treated with both TNF-α and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction (P<0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction (P<0.05). Conclusion Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF-α stimulation.